Ion channels are cellular proteins that regulate the flow of ions, including calcium, potassium, sodium and chloride into and out of cells. These channels are present in all human cells and affect such physiological processes as nerve transmission, muscle contraction, cellular secretion, regulation of heartbeat, dilation of arteries, release of insulin, and regulation of renal electrolyte transport. Among the ion channels, potassium channels are the most ubiquitous and diverse, being found in a variety of animal cells such as nervous, muscular, glandular, immune, reproductive, and epithelial tissue. These channels allow the flow of potassium in and/or out of the cell under certain conditions. For example, the outward flow of potassium ions upon opening of these channels makes the interior of the cell more negative, counteracting depolarizing voltages applied to the cell. These channels are regulated, e.g., by calcium sensitivity, voltage-gating, second messengers, extracellular ligands, and ATP-sensitivity.
Potassium channels are made by alpha subunits that fall into at least 8 families, based on predicted structural and functional similarities (Wei et al., Neuropharmacology 35(7): 805–829 (1997)). Three of these families (Kv, eag-related, and KQT) share a common motif of six transmembrane domains and are primarily gated by voltage. Two other families/ also contain this motif but are gated by cyclic nucleotides (CNG) and calcium (small conductance and intermediate conductance potassium channels), respectively. The small conductance and intermediate conductance, calcium activated potassium channels comprise a family of calcium activated potassium channels gated solely by calcium, with a unit conductance of 2-20 and 20-85 pS, respectively. Macroscopic and unitary intermediate conductance, calcium activated potassium channel currents show inward rectification (see, e.g., Ishii et al., Proc. Natl. Acad. Sci USA 94: 11651–11656 (1997). The three other families of potassium channel alpha subunits have distinct patterns of transmembrane domains. Slo family potassium channels, or BK channels have seven transmembrane domains (Meera et al., Proc. Natl. Acad. Sci. U.S.A. 94(25): 14066–71 (1997)) and are gated by both voltage and calcium or pH (Schreiber et al., J. Biol. Chem. 273: 3509–16 (1998)). Another family, the inward rectifier potassium channels (Kir), belongs to a structural family containing two transmembrane domains, and an eighth functionally diverse family (TP, or “two-pore”) contains two tandem repeats of this inward rectifier motif.
Potassium channels are typically formed by four alpha subunits, and can be homomeric (made of identical alpha subunits) or heteromeric (made of two or more distinct types of alpha subunits). In addition, potassium channels made from Kv, KQT and Slo or BK subunits have often been found to contain additional, structurally distinct auxiliary, or beta, subunits. These subunits do not form potassium channels themselves, but instead they act as auxiliary subunits to modify the functional properties of channels formed by alpha subunits. For example, the Kv beta subunits are cytoplasmic and are known to increase the surface expression of Kv channels and/or modify inactivation kinetics of the channel (Heinemann et al., J. Physiol. 493: 625–633 (1996); Shi et al., Neuron 16(4): 843–852 (1996)). In another example, the KQT family beta subunit, minK, primarily changes activation kinetics (Sanguinetti et al., Nature 384: 80–83 (1996)).
The intermediate conductance, calcium activated potassium channel is also called SK4, KCa4, IKCa, SMIK, and Gardos. Intermediate conductance, calcium activated potassium channels have been previously described in the literature by their electrophysiology. For example, the Gardos channel, a well-known intermediate conductance, calcium activated potassium channel, is opened by submicromolar concentrations of internal calcium and has a rectifying unit conductance, ranging from 50 pS at −120 mV to 13 pS at 120 mV (symmetrical 120 mM K+; Christopherson, J. Membrane Biol. 119: 75–83 (1991)). Intermediate conductance, calcium activated potassium channels are blocked by charybdotoxin (CTX) but not the structurally related peptide iberiotoxin (IBX), both of which block BK channels (Brugnara et al., J. Membr. Biol. 147: 71–82 (1995)). Intermediate conductance, calcium activated potassium channels are also blocked by maurotoxin. Apamin, a potent blocker of certain native (Vincent et al., J. Biochem. 14: 2521 (1975); Blatz & Magleby, Nature 323: 718–720 (1986)) and cloned SK channels does not block intermediate conductance, calcium activated potassium channels (de-Allie et al., Br. J. Pharm. 117: 479–487 (1996)). The Gardos channel is also blocked by some imidazole compounds, such as clotrimazole, but not ketoconazole (Brugnara et al, J. Clin. Invest., 92: 520–526 (1993)). Intermediate conductance, calcium activated potassium channels can therefore be distinguished from the other calcium activated potassium channels by their biophysical and pharmacological profiles. Intermediate conductance, calcium activated potassium channels from different tissues have been reported to possess a wide range of unit conductance values.
Human intermediate conductance, calcium activated potassium channels have been cloned and characterized (see, e.g., Ishii et al., Proc. Natl. Acad. Sci. USA 94: 11651–11656 (1997); Genbank Accession No. AF0225150; Joiner et al., Proc. Natl. Acad. Sci. USA 94: 11013–11018 (1997); Genbank Accession No. AF000972; Lodsdon et al., J. Biol. Chem. 272: 32723–32726 (1997); Genbank Accession No. AF022797; and Jensen et al., Am. J. Physiol. 275: C848–856 (1998); see also WO 98/11139; WO 99/03882; WO 99/25347; and WO 00/12711). Non-human intermediate conductance, calcium activated potassium channels have also been cloned, e.g., from mouse and rats (see, e.g., Vandorpe et al., J. Biol. Chem. 273: 21542–21553 (1998); Genbank Accession No. NM—032397; Warth et al., Pflugers Arch. 438: 437–444 (1999); Genbank Accession No. AJ133438; and Neylon et al., Circ. Res. (online)85: E33–E43 (1999); Genbank Accession No. AF190458). The gene for the intermediate conductance, calcium activated potassium channels is named KCNN4 and it is located on chromosome 19q13.2 (Ghanshani et al., Genomics 51: 160–161 (1998)).
The intermediate conductance, calcium activated potassium channel is implicated in the regulation of mammalian cell proliferation (see, for example, Wulff et al., Proc. Nat. Acad. Sci. USA 97: 8151–8156 (2000)) and the dehydration and sickling of erythrocytes in sickle cell disease. Sickle cell disease has been recognized within West Africa for several centuries. Sickle cell anemia and the existence of sickle hemoglobin (Hb S) was the first genetic disease to be understood at the molecular level. It is recognized today as the morphological and clinical result of a glycine to valine substitution at the No. 6 position of the beta-globin chain (Ingram, Nature 178: 792–794 (1956)). The origin of the amino acid change and of the disease state is the consequence of a single nucleotide substitution (Marotta et al., J. Biol. Chem. 252: 5040–5053 (1977)).
Normal erythrocytes are comprised of approximately 70% water. Water crosses a normal erythrocyte membrane in milliseconds. Loss of cell water causes an exponential increase in cytoplasmic viscosity as the mean cell hemoglobin concentration (MCHC) rises above about 32 g/dl. Since cytoplasmic viscosity is a major determinate of erythrocyte deformability and sickling, the dehydration of the erythrocyte has substantial rheological and pathological consequences. Regulation of erythrocyte dehydration is recognized as an important therapeutic approach for treating sickle cell disease. Since cell water follows any osmotic change in intracellular ion concentration, maintaining the red cell's potassium concentration is of particular importance (Stuart et al., Brit J. Haematol. 69: 1–4 (1988)).
An approach towards therapeutically treating dehydrated sickle cells involves altering erythrocyte potassium flux by targeting a calcium-dependent potassium channel. This calcium activated potassium channel is also referred to as the Gardos channel (Brugnara et al, J. Clin. Invest. 92: 520–526 (1993)). Recently, a cloned human intermediate conductance, calcium activated potassium channel, was shown to be substantially similar to the Gardos channel in terms of both its biophysical and pharmacological properties (Ishii et al., Proc. Natl. Acad. Sci. USA 94: 11651–11656 (1997)).
In vitro studies have shown that clotrimazole, an imidazole-containing antimycotic agent, blocks Ca2+-activated K+ flux and cell dehydration in sickle erythrocytes (Brugnara et al., J. Clin. Invest. 92: 520–526 (1993)). Studies in a transgenic mouse model for sickle cell disease, SAD-1 mouse (Trudel et al., EMBO J. 11: 3157–3165 (1991)), show that oral administration of clotrimazole leads to inhibition of the red cell Gardos channel, increased red cell K+ content, a decreased mean corpuscular hemoglobin concentration (MCHC) and decreased cell density (De Franceschi et al., J. Clin. Invest. 93: 1670–1676 (1994)). Moreover, therapy with oral clotrimazole induces inhibition of the Gardos channel and reduces erythrocyte dehydration in patients with sickle cell disease (Brugnara et al., J. Clin. Invest. 97: 1227–1234 (1996)). Other antimycotic agents, which inhibit the Gardos channel in vitro, include miconazole, econazole, butoconazole, oxiconazole and sulconazole (U.S. Pat. No. 5,273,992 to Brugnara et al.). All of these compounds contain an imidazole-like ring. i.e., a heteroaryl ring containing two or more nitrogens.
Although of demonstrable efficacy, the imidazole-based Gardos channel inhibitors that have been explored to date are hampered by several shortcomings including a well-documented potential for hepatotoxicity. This toxicity is exacerbated by the inhibitors' low potencies, non-specific interactions with potassium channels other than the Gardos channel and low bioavailabilities, each of which motivate for the administration of higher and more frequent dosages of the inhibitors.
Glaucoma is a disease characterized by increased intraocular pressure. Increased intraocular pressure is associated with many diseases including, but not limited to, primary open-angle glaucoma, normal tension glaucoma, angle-closure glaucoma, acute glaucoma, pigmentary glaucoma, neovascular glaucoma, or trauma related glaucoma, Sturge-Weber syndrome, uveitis, and exfoliation syndrome.
Currently, there are a variety of drugs available that employ different mechanisms to lower intraocular pressure, e.g., timolol, betaxolol, levobunolol, acetazolamide, methazolamide, dichlorphenamide, dorzolamide, brinzolamide, latanoprost, brimonidine, and rescula (see, e.g., U.S. Pat. Nos. 6,172,054, 6,172,109, and 5,652,236). Miotics, beta blockers, alpha-2 agonists, carbonic anhydrase inhibitors, beta adrenergic blockers, prostaglandins and docosanoid are all currently used alone or in combination to treat glaucoma. Miotics and prostaglandins are believed to lower intraocular pressure by increasing drainage of the intraocular fluid, while beta blockers, alpha-2 agonists and carbonic anhydrase are believed to lower intraocular pressure by decreasing production of intraocular fluid thereby reducing the flow of fluid into the eye. All are characterized by side effects ranging from red eye and blurring of vision to decreased blood pressure and breathing difficulties.
In view of the above-described shortcomings of currently known methods of treating diseases in which the intermediate conductance, calcium activated potassium channel is implicated, a substantial advance in the treatment of diseases related to potassium flux is expected from the discovery of new intermediate conductance, calcium activated potassium channel inhibitors. The present invention provides a new genus of such ion channel inhibitors based on a sulfonamide-containing scaffold.